In the past few years there has been the development of numerous "immunotoxins" which consist of a cell type specific targeting antibody linked to a cytotoxic agent. The antibody generally recognizes a plasma membrane antigen to which the immunotoxin binds. After the immunotoxin is bound to the cell surface, it is endocytosed into the cell, where it interferes with cellular biochemical reactions, generally protein synthesis, thereby causing cell death. Thus, immunotoxins have three key components, the cell targeting antibody molecule, the covalent bond which holds antibody and toxin together, and the cytotoxic agent. Vitetta et al. Science, 238:1098 (1987).
Most often the antibody portion of an immunotoxin consists of a monoclonal antibody. Kohler and Milstein, Nature (London) 256:495 (1975). Monoclonal antibodies are chemically homogenous, and thus facilitate the construction of truly cell type specific immunotoxins. Immunotoxins can, however, be constructed with polyclonal antibodies.
A variety of molecules have been used for the toxin portion of the conjugate, including alkylating and anoimetabolic agents, as well as alkaloids and various toxic proteins or peptides and portions or fragments of toxins. The latter molecules (toxins) are generally produced by bacteria or plants and exert their cytoxic effect by enzymatically interrupting protein synthesis at the ribosomal level. Enzymatically active toxin and fragments thereof are often preferred, and are exemplied by diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (Pseudomonas aeruginosa), abrin A chain, and ricin A chain.
Binding of antibody to the toxin is effected by a covalent linkage, the nature of which varies depending on the type of toxin employed. For protein or peptide toxins, generally disulfide bond is used to link antibody to the toxin. This type of linkage has been widely used because it is generally believed that the cytotoxic moiety must be released from the antibody after entry into the cell for the toxin to exert its effect. A disulfide link between antibody and toxin is served by the reducing environment inside the cell. Examples of reagents that have been used to from the disulfide bond include N-succinimidyl-3-(2-pyridyl- dithio(propionate) (SPDP), and 2-iminothiolane hydrochloride (2-IT).
One of the drawbacks associated with the use of a disulfide bond is that the bond is cleaved in the extra cellular circulation. Blakey et al. Cancer Research, 47:947 (1987); Levin et al., J. Clin. Invest., 77:977 (1986). For example, it has been shown that a significant fraction of immunotoxin is cleaved in the blood within eight hours of intravenous administration, and most of the conjugate is cleaved within 24 hours. Thus, the amount of immunotoxin available for reaction with the target cell is not as great as may be desirable, and, moreover, free antibody may compete with the immunotoxin for binding to the target cell thus reducing the efficacy of treatment. In addition, toxicity and other pharmacological preparation may be effected by the release of the toxin moiety.
Several patents show chemicals that have been, and continue to be utilized for forming disulfide cross-linked immunotoxins. U.S. Pat. Nos. 4,350,626 and 4,450,154 claim immunotoxins in which a monovalent (Fab), or divalent (Fab'), fragment of antibody having tumor cell type specificity is coupled to recin A chain. Coupling was affected through cysteine-thiols on the two proteins, with or without an intervening bifunctional crosslinking group. Moreover, U.S. Pat. Nos. 4,357,273 and 4,638,049 describe similar immunotoxins wherein diphtheria toxin was conjugated in lieu of ricin A chain. U.S. Pat. No. 4,534,211 discloses conjugates wherein the cytotoxic moiety is attached to the antibody by at least one sulphur atom. Lastly, U.S. Pat. No. 4,340,535 shows antibody-ricin A conjugates wherein the disulfide bond is effected by SPDP.
In addition to disulfide bonds, antibody and toxins have also been joined by other chemical bonds. One construct makes use of an acid-labile crosslink. The acid-labile linkage is served when the conjugate is taken up by a cell and compartmentalized into a cellular compartment having a pH value which is lower than that outside the cell. Blatter et al., BioChem., 24:1517 (1985). A second means of crosslinking antibody and toxin has been to use a peptide bond with an amino acid sequence that is thought to be recognized by proteases found in the cell's cytoplasm. U.S. Pat. No. 4,571,958. Neither of these crosslinkers are favored in constructing immunotoxins as either they breakdown more rapidly than the disulfide bond crosslinkers in blood, or else the linkage is not readily served once the conjugate has entered the cytoplasm.
Finally, there have been several reports wherein antibody and cytotoxin are linked by a thioether covalent bond. Wawrzynczak E., and Thorpe, P. (1987), Methods for Preparing Immunotoxins: Effect of the Linkage on Activity and Stability. In Immunoconjugates, Antibody Conjugates in Radioimaging and Therapy of Cancer (C-W., Vogel, ed), New York, Oxford University Press, pp. 28-55. These conjugates were developed to obviate the undesirable cleavage of antibody and toxin which occurs in the extra cellular circulation associated with disulfide linked conjugates. Thioether linked immunotoxins are, however, considerably less active than disulfide linked immunotoxins. For example, thioether conjugates exhibit 70% less protein synthesis inhibitory activity in vitro assays, and are 99% less cytotoxic compared to analogous disulfide conjugates. The loss of efficacy is throught to result, at least in part, from the inability of toxin to separate from antibody once the conjugate is taken up inside the cell, a putative requirement for cytotoxity. For example, it has been stated by Fulton et al., that "If the bond between antibody and A chain is not reducible, then toxicity is virtually abolished", Fulton, R. et al., (1987), Immunotoxins containing ricin A and B chains: In Immunoconjugates, Antibody Conjugates in Radioimaging and Therapy of Cancer (C. W. Vogel, ed), New York, Oxford University Press, pp. 56-70. Consequently, despite their extra cellular stability thioether bonds are disfavored to form immunotoxin conjugates.
It should be apparent from the foregoing discussion that the preferred linkage between antibody and toxin should be stable in the extra cellular circulation, and highly cytotoxic after it is endocytosed by the target cell.